protein gel electrophoresis
DESCRIPTION
Protein Gel Electrophoresis. Native PAGE Native Gradient PAGE Urea PAGE SDS PAGE SDS Gradient PAGE IEF 2D PAGE Western Blot. Principle. Proteins move in the electric field. Their relative speed depends on the charge, size, and shape of the protein. From large to small and simple. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Protein Gel Electrophoresis](https://reader035.vdocuments.site/reader035/viewer/2022062411/56816953550346895de0fc01/html5/thumbnails/1.jpg)
Protein Gel Electrophoresis
1. Native PAGE
2. Native Gradient PAGE
3. Urea PAGE
4. SDS PAGE5. SDS Gradient PAGE
6. IEF
7. 2D PAGE
8. Western Blot
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Principle
Proteins move in the electric field. Their relative speed depends on the charge, size, and shape of the protein
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From large to small and simple
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Protein visualization on gels
Immediately after electrophoresis proteins in the gels are precipitated by either adding alcohol containing solutions or strong acids (e.g. TCA).
Protein are often stained by Coomassie Blue dye or by photography-like treatment with AgNO3 (silver staining)
There are many other stains available (e.g. Stains-all, fluorescence probes etc.)
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Example of silver stained gel
Silver staining is usually 10-100 times more sensitive than Coomassie Blue staining, but it is more complicated.
Faint but still visible bands on this gel contain less than 0.5 ng of protein!
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Native PAGE
Useful for:
1. Examining protein-protein protein-ligand interactions
2. Detecting protein isoforms/conformers
Separates folded proteins and protein-protein or protein-ligand complexes by charge, size, and shape
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Native PAGE examples
In the absence of phospholipids, both twinfilins run as a single sharp band on this gel. PI(4,5)P2 causes twinfilin-1 and twinfilin-2 to move more rapidly toward the anode, indicating a net increase in the negative charge and thus a binding interaction.
Dimerization of KIR2DL1 in the presence of Co2
Vartiainen et al. JBC 2003 Qing R. Fan et al. JBC 2000
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Native gradient PAGE
Separate native proteins by size – proteins stop moving when they reach a sertain gel density (but this may take a very long time ...)
A great technique to study protien oligomerization!
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Native gradient PAGE example
Zavialov et al. Mol. Microbiol. 2002
Native 4-15% gradient PAGE
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Urea PAGE
Separates denatured proteins by size/charge
Typically 6-8 M urea is added into the gel
A great technique to study protein modifications!
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Example of Urea PAGE
Urea PAGE of samples of heat shock protein 25
Zavialov et al. BBA 1998
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SDS PAGE
Due to high density of binding of SDS to proteins, the ratio size/charge is nearly the same for many SDS denatured proteins. Hence proteins are separated only by length of their polypeptide chains (but not by differences in charge).
Great separation. Allows estimation of the size of polypeptide chains
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SDS gradient PAGE
Bands in SDS gradient gel are usually sharper than in homogeneous SDS PAGE
5-20% SDS PAGE
12.5% SDS PAGE
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SDS-PAGESodium Dodecyl Sulfate -
Polyacrylamid Gel Electrophoresis
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Sodium Dodecyl Sulfate• SDS is a common ingredient in detergents• Other names for SDS include laurel sulfate
and sodium laurel sulfate• As a detergent SDS destroys protein
secondary, tertiary and quaternary structure• This makes proteins rod shaped• SDS also sticks to proteins in a ratio of
approximately 1.4 g of SDS for each gram of protein
• Negative charge on the sulfate groups of SDS mask any charge on the protein
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PolarHydrophilic head
Non-polarHydrophobic tail
SDSSodium Dodecyl Sulfate
• Because it is amphipathic, SDS is a potent detergent
H-C-C-C-C-C-C-C-C-C-C-C-C-O-S-O-Na+
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
C12H25NaO4
S
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SDS and Proteins
SDS
Protein
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SDS and Proteins
In aqueous solutions, SDS polarizes releasing Na+ and retaining a negative charge on the sulfate head
So much SDS binds to proteins that the negative charge on the SDS drowns out any net charge on protein side chains
In the presence of SDS all proteins have uniform shape and charge per unit length
SDS nonpolar chains arrange themselves on proteins and destroy secondary tertiary and quarternary structrure
Thus shape is no longer an issue as the protein SDS complex becomes rod shaped
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Acrylamide
Acrylamide
Polyacrylamide Gels• Polyacrilamide is a polymer made of
acrylamide (C3H5NO) and bis-acrilamide (N,N’-methylene-bis-acrylamide C7H10N2O2)
O
CH
CH2
NH2C
O
CHCH2
NH2C
CH2
bis-Acrylamide
O
CHCH2
NH2C
Acrylamide
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Polyacrylamide Gels
O
CHCH2
NH2C
O
CHCH2
NH2C
SO4-.
• Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate
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Polyacrylamide Gels1. Acrylamide polymerizes in the presence of free
radicals typically supplied by ammonium persulfate
SO4-.
O
CHCH2
NH2C
O
CHCH2
NH2CNH2
O
CHCH2
C
O
CHCH2
NH2C
2.TMED (N,N,N’,N’-tetramethylethylenediamine) serves as a catalyst in the reaction
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Polyacrylamide Gels• bis-Acrylamide polymerizes along with
acrylamide forming cross-links between acrylamide chainsO
CHCH2
NH2C
O
CHCH2
NH2C
O
CHCH2
NH2CNH2
O
CHCH2
C
O
CHCH2
NH2C
O
CHCH2
NH2C
bis-Acrylamide
O
CH
CH2
NH2C
O
CHCH2
NH2C
CH2
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Polyacrylamide Gels• bis-Acrylamide polymerizes along with
acrylamide forming cross-links between acrylamide chains
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Polyacrylamide Gels• Pore size in gels can be varied by varying the
ratio of acrylamide to bis-acrylamide
Lots of bis-acrylamideLittle bis-acrylamide
Protein separations typically use a 29:1 or 37.5:1 acrylamide to bis ratio
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1 2 3
SDS-PAGE
Addition of SDS23
1 Protein becomes rod-shaped with uniform charge distribution
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IEF
Separates proteins by their isoelectric points (pI)
Each protein has own pI = pH at which the protein has equal amount of positive and negative charges (the net charge is zero)
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IEFMixtures of ampholytes, small amphoteric molecules with high buffering capacity near their pI, are used to generate the pH gradient.
Positively and negatively charged proteins move to – and +, respectively, until they reach pI.
PI of proteins can be theoretically predicted. Therefore, IEF can also be used for protein identification.
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IEF example
Zavialov A.
IEF 4-6.5 pH gradient
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2D PAGE
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2D PAGE
Lung V79 cells from chinese hamster
Guillermo Senisterra Dept. of Physics-University of Waterloo-Waterloo-Ontario N2L 3G1-Canada
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Proteomics Pathway
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Proteomics Pathway
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Western Blotting (WB)WB is a protein detection technique that combines the separation power of SDS PAGE together with high recognition specificity of antibodies
An antibody against the target protein could be purified from serum of animals (mice, rabbits, goats) immunized with this protein
Alternatively, if protein contains a commonly used tag or epitope, an antibody against the tag/epitope could be purchase from a commercial source (e.g. anti-6 His antibody)
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WB: 4 steps
1. Separation of proteins using SDS PAGE
2. Transfer of the proteins onto e.g. a nitrocellulose membrane (blotting)
3. Immune reactions
4. Visualization
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WB, Step 2: Blotting
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WB, Steps 3-4: Detection